Natural killer (NK) cells are a subset of large granular lymphocytes that act as cytotoxic immune cells. The cytotoxic activity mediated by NK cells naturally against target cells (e.g., cancer cells, virally infected cells) is generally expressed a being the result of a “balance” of positive and negative signals transmitted respectively by activating and inhibitory cell surface receptors.
NK cells can be identified by any number of known cell surface markers which vary between species (e.g., in humans CD56, CD16, NKp44, NKp46, and NKp30 are often used; in mice NK1.1, Ly49A-W, CD49b are often used). In an active state, NK cells are capable of killing certain autologous, allogeneic, and even xenogeneic tumor cells, virus-infected cells, certain bacteria (e.g., Salmonella typhi), and other target cells. NK cells appear to preferentially kill target cells that express little or no Major Histocompatibility Class I (MHCI or MHC-I) molecules on their surface. NK cells also kill target cells to which antibody molecules have attached, a mechanism known as antibody-dependent cellular cytotoxicity (ADCC). In action against target cells, NK cells can release pore-forming proteins called perforins, proteolytic enzymes called granzymes, and cytokines/chemokines (e.g., TNFα, IFNγ) that directly lead to target cell apoptosis or lysis, or that regulate other immune responses. Upon activation, NK cells also may express Fas ligand (FasL), enabling these cells to induce apoptosis in cells that express Fas.
Sufficient NK cell activity and NK cell count typically are both necessary to mounting an adequate NK cell-mediated immune response. NK cells may be present in normal numbers in an individual, but if not activated these cells will be ineffective in performing vital immune system functions, such as eliminating abnormal cells. Decreased NK cell activity is linked to the development and progression of many diseases. For example, research has demonstrated that low NK cell activity causes greater susceptibility to diseases such as chronic fatigue syndrome (CFS), viral infections, and the development of cancers.
NK cell activity is regulated by NK cell activity-modulating receptors (NKCAMRs), which may be specific for various ligands such as MHC-I molecules, MHC-I homologs, or other biological molecules expressed on target cells. NK cells in an individual typically present a number of activating and inhibitory receptors. The activity of NK cells is regulated by a balance of signals transduced through these activating and inhibitory receptors. Most NK cell activity-modulating receptors appear to belong to one of two classes of proteins: the immunoglobulin (Ig)-like receptor superfamily (IgSF) or the C-type lectin-like receptor (CTLR) super family. See, e.g., Radaev and Sun (2003) Annu. Rev. Biomol. Struct. 32: 93-114). However, other forms of NKCAMRs are known.
Many NK cell activating receptors belong to the Ig superfamily (IgSF) (such receptors also may be referred to as Ig-like receptors or “ILRs” herein). Activating ILR NK receptors (AILRs) include, e.g., CD2, CD16, CD69, DNAX accessory molecule-1 (DNAM-1), 2B4, NK1.1; killer immunoglobulin (Ig)-like activating receptors (KARs); ILTs/LIRs; and natural cytotoxicity receptors (NCRs), such as NKp44, NKp46, and NKp30. Several other activating receptors belong to the CLTR superfamily (e.g., NKRP-1, CD69; CD94/NKG2C and CD94/NKG2E heterodimers, NKG2D homodimer, and in mice, activating isoforms of Ly49, such as Ly49A-D). Still other activating receptors (e.g., LFA-1 and VLA-4) belong to the integrin protein superfamily and other activating receptors may have even other distinguishable structures. Many activating receptors possess extracellular domains that bind to MHC-I molecules, and cytoplasmic domains that are relatively short and lack the immunoreceptor tyrosine-based inhibition motif (ITIM) signaling motifs characteristic of inhibitory NK receptors. The transmembrane domains of these receptors typically include a charged amino acid residue that facilitates their association with signal transduction-associated molecules, e.g., CD3zeta, FcεRIγ, DAP12, and DAP10 (2B4, however, appears to be an exception to this general rule), which contain short amino acid sequences termed an “immunoreceptor tyrosine-based activating motif” (ITAMs) that propagate NK cell-activating signals. Receptor 2B4 contains 4 Immunoreceptor Tyrosine-based Switch Motifs (ITSMs) in its cytoplasmic tail. ITSM motifs can also be found in NKCARs CS1/CRACC and NTB-A. The cytoplasmic domains of 2B4 and SLAM contain two or more unique tyrosine-based motifs that resemble motifs presents in activating and inhibitory receptors and can recruit the SH2-domain containing proteins SHP-2 and SLAM-associated protein (SAP).
Stress-induced molecules, e.g., MIC-A, MIC-B, and ULBPs (in humans), and Rae-1 and H-60 (in mice), can serve as ligands for activating receptors, such as the NKG2D homodimer. Cellular carbohydrates, pathogenic antigens, and antibodies can also be activating receptors ligands. For example, NKR-P1 may bind to carbohydrate ligands and trigger NK cell activation, particularly against tumor cells which exhibit aberrant glycosylation patterns. Viral hemagglutinins may serve as ligands for natural cytotoxic receptors (NCRs), such as ILR NKCARs NKp30, NKp44, NKp46, and NKp80.
Activating receptors can either directly transduce activating signals or can act in connection with adaptor molecules or other receptors, either in the context of a coordinated response between receptors that are sometimes singularly effective or in the context of coreceptor-receptor pairings. For example, NCRs typically lack ITAMs and, accordingly, bind to adaptor molecules through a charged residue in their transmembrane domains (e.g., NKp30 associates with the CD3 zeta chain; NKp44 associates with DAP12 and/or KARAP; NKp46 is coupled to the CD3 zeta chain and FcRIγ chain), which are, in turn, able to recruit protein tyrosine kinases (PTKs) in order to propagate NK cell-activating signals. CD16, which is an activating receptor important to NK cell-mediated ADCC and cytokine production, associates with homodimers or heterodimers formed of CD3 zeta and/or gamma chains. NKG2D appears to play a complementary and/or synergistic role with NCRs and activating receptors in NK cell activation. Activation of NK cells against particular targets may require coordinated activation of multiple activating receptors or NCRs, or only action of a single receptor. Other triggering surface molecules including 2B4 and NKp80 appear to function as coreceptors for NK cell activation.
Activating isoforms of human killer immunoglobulin-like receptors (KIRs) (e.g., KIR2DS and KIR3DS) and murine Ly-49 proteins (e.g., Ly-49D and Ly-49H) are expressed by some NK cells. Stimulation or tolerance of natural killer (NK) cells is achieved through a cross-talk of signals derived from cell surface activating and inhibitory receptors. Killer cell immunoglobulin-like receptors (KIR) are a family of highly polymorphic activating and inhibitory receptors that serve as key regulators of human NK cell function. Distinct structural domains in different KIR family members determine function by providing docking sites for ligands or signalling proteins. See Campbell & Purdy (2011) Immunology 132(3): 315-25. These molecules differ from their inhibitory counterparts, which are discussed below, by lacking ITIMs in their relatively shorter cytoplasmic domains, and possessing a charged transmembrane region that associates with signal-transducing polypeptides, such as disulfide-linked dimers of DAP12.
ILR (IgSF) NK cell inhibitory receptors include a number of different human KIRs specific for HLA-A, -B, or -C allotypes, KIRs may recognize multiple alleles within a particular allotype, e.g., KIR2DL1 recognizes HLA-Cw2, Cw4, and Cw6 allotypes. CTLR superfamily inhibitory receptors include members of the CD94/NKG2 protein family, which comprise receptors formed by lectin-like CD94 with various members of the NKG2 family, such as NKG2A, and recognize the nonclassical MCH-I molecules HLA-E and Qa-1 (in humans and mice, respectively), and the murine Ly49 molecules that recognize the classical MHC-I molecules in mice. In even further contrast, NKRP1A, Nkrp1f and Nkrp1d are inhibitory receptors whose ligands are not MHC-related, but are CTLR family members expressed on various cell types, such as dendritic cells, macrophages, and lymphocytes.
MHC class I-specific NKCIRs include CTLR Ly-49 receptors (in mice); the IgSF receptors Leukocyte Immunoglobulin-like Receptor (LIRs) (in humans), KIRs (e.g., p58 and p70 Killer-cell Immunoglobulin-like Receptors) (in humans), and CTLR CD94/NKG2 receptors (in mice and humans). All MHC-1-specific NKCIRs appear to use a common inhibitory mechanism apparently involving phosphorylation of ITIMs in their cytoplasmic domains in the course of MHC-I binding, and recruitment of tyrosine phosphatases (e.g., SHP-1 and SHP-2) to the phosphorylated ITIMs, resulting in the inhibition of proximal protein tyrosine kinases (PTKs) involved in NK activation through NKCARs. Antibodies against activity-modulating receptors, such as KIR, have been previously described. There also has been at least some suggestion of combining anti-NK receptor antibodies, such as anti-KIR antibodies, with other anti-cancer agents in the prior art. For example, WO 2004/056392 describes anti-NKp30 and/or anti-NKp46 antibodies used in admixture with interleukin-2 (IL-2). WO 2008/084106 describes anti-KIR formulations, dosages and dose regimens. WO 2005/079766 also describes combinations of antibodies (e.g., anti-tissue factor antibodies) including anti-KIR antibodies for use in cancer therapies. WO 2005/003168 and WO 2005/003172 describe combinations of a number of anti-KIR antibodies with a variety of agents, including IL-2 and interleukin-21 (IL-21). WO 2005/037306 similarly describes combinations of IL-21, IL-21 derivatives, and IL-21 analogues in combination with anti-KIR antibodies. WO 2005/009465 describes the combination of a therapeutic antibody (e.g., Rituxan) in combination with a compound that blocks an inhibitory receptor or stimulates an activating receptor of an NK cell (e.g., an anti-KIR monoclonal antibody, such as the monoclonal antibody DF200, or an anti-NKp30 monoclonal antibody) in order to enhance the efficiency of the treatment with therapeutic antibodies in human subjects.
Autoimmune Disease
An autoimmune disorder is a condition that occurs when the immune system mistakenly attacks and destroys healthy body tissue. There are more than 80 different types of autoimmune disorders. Normally the immune system's white blood cells help protect the body from harmful substances, called antigens. Examples of antigens include bacteria, viruses, toxins, cancer cells, and blood or tissues from another person or species. The immune system produces antibodies that destroy these harmful substances.
However, in patients with an autoimmune disorder, the immune system can not distinguish between self and non-self (e.g., healthy tissue and foreign antigens). The result is an immune response that destroys normal body tissues. This response is a hypersensitivity reaction similar to the response in allergic conditions.
In allergies, the immune system reacts to an outside substance that it normally would ignore. With autoimmune disorders, the immune system reacts to normal body tissues that it would normally ignore.
What causes the immune system to no longer tell the difference between healthy body tissues and antigens is unknown. One theory is that some microorganisms (such as bacteria or viruses) or drugs may trigger some of these changes, especially in people who have genes that make them more likely to get autoimmune disorders.
An autoimmune disorder may result in the destruction of one or more types of body tissue, abnormal growth of an organ, and changes in organ function. An autoimmune disorder may affect one or more organ or tissue types. Organs and tissues commonly affected by autoimmune disorders include blood vessels, connective tissues, endocrine glands (e.g., thyroid or pancreas), joints, muscles, red blood cells, and skin. A person may have more than one autoimmune disorder at the same time.
Symptoms of an autoimmune disease vary based on the disease and location of the abnormal immune response. Common symptoms that often occur with autoimmune diseases include fatigue, fever, and a general ill-feeling (malaise). Tests that may be done to diagnose an autoimmune disorder may include: antinuclear antibody tests, autoantibody tests, CBC, C-reactive protein (CRP), and erythrocyte sedimentation rate (ESR).
Medicines are often prescribed to control or reduce the immune system's response. They are often called immunosuppressive medicines. Such medicines may include corticosteroids (such as prednisone) and nonsteroid drugs such as azathioprine, cyclophosphamide, mycophenolate, sirolimus, or tacrolimus.
Complications are common and depend on the disease. Side effects of medications used to suppress the immune system can be severe, such as infections that can be hard to control. “Autoimmune disorders.” MedlinePlus—U.S. National Library of Medicine (Apr. 19, 2012).
Inflammatory Conditions
Inflammation is part of the complex biological response of vascular tissues to harmful stimuli, such as pathogens, damaged cells, or irritants. Inflammation is a protective attempt by the organism to remove the injurious stimuli and to initiate the healing process. Without inflammation, wounds and infections would never heal. Similarly, progressive destruction of the tissue would compromise the survival of the organism. However, chronic inflammation can also lead to a host of diseases, such as hay fever, periodontitis, atherosclerosis, rheumatoid arthritis, and even cancer (e.g., gallbladder carcinoma). It is for that reason that inflammation is normally closely regulated by the body.
Inflammation can be classified as either acute or chronic. Acute inflammation is the initial response of the body to harmful stimuli and is achieved by the increased movement of plasma and leukocytes (especially granulocytes) from the blood into the injured tissues. A cascade of biochemical events propagates and matures the inflammatory response, involving the local vascular system, the immune system, and various cells within the injured tissue. Prolonged inflammation, known as chronic inflammation, leads to a progressive shift in the type of cells present at the site of inflammation and is characterized by simultaneous destruction and healing of the tissue from the inflammatory process. Kindt, et al. (2006) Kuby Immunology [6th Ed.]
T-cells are involved in the promulgation of inflammation. Differentiation of naïve T cells leads to the generation of T-cell subsets, each possessing distinct cytokine expression profiles for serving different immune functions. Through the activation of separate signaling pathways, this process results in both differentiated helper T (Th) cells, termed Th1, Th2 and Th17, and induced regulatory T cells, which suppress Th cells. These different cells are important for combating infectious diseases and cancers; however, when aberrant, they can be responsible for chronic inflammatory diseases. One such disease is inflammatory bowel disease (IBD), in which each T-cell subset can have a role in disease. Zenewicz, et al. (2009) Trends in Molecular Medicine 15(5): 199-207.
While NK cells have received a great deal of attention in the scientific literature for their potential contribution to anti-tumor and anti-viral responses, few studies have been directed to examining the role of NK cells in inflammation and autoimmunity, particularly the KIR2DL1, 2 and/or 3-expressing subsets. The approach toward these NK cells, if anything, has been to seek to eliminate or inhibit NK cells on the basis that they may contribute to inflammation and autoimmunity. The effect of KIR2DL1, 2 and/or 3-mediated potentiating of NK cell cytotoxicity in inflammatory settings has to date not been addressed.
Consequently, there is a need in the art for methods of using NK cell modulation to provide improved benefit to patients.